rasz protein of saccharomyces cereuisiae is methyl ... · the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 264, No . 20, Issue of July 15, PP. 1186-11673,1989 Printed in U.S.A.

RASZ Protein of Saccharomyces cereuisiae Is Methyl-esterified at Its Carboxyl Terminus*

(Received for publication, February 9, 1989)

Robert J. DeschenesS, Julie B. Stimmelell, Steven Clarke$, Jeff Stock[[, and James R. Broach From the Department of Biology, Princeton University, Princeton, New Jersey 08544 and the §Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, California 90024

Yeast and mammalian RAS gene products are GTP- binding proteins that are posttranslationally localized to the inner surface of the plasma membrane. This localization is accomplished by the addition of a lipid moiety to a conserved cysteine residue close to the carboxyl terminus. In a previous report we showed that the mammalian Ha-ras protein is also modified posttranslationally by methyl esterification. Here we show that the yeast RAS2 protein is posttranslationally modified by methyl esterification at or near the carboxyl terminus. We also present evidence indicating that the methyl ester is linked to the conserved cysteine residue, implying that RAS2 protein is cleaved to ex- pose this cysteine as the carboxyl-terminal residue. This maturation pathway may be shared by a family of proteins that are initially synthesized as soluble proteins and must become membrane-localized to function.

Mammalian ras protooncogenes encode a family of membrane-associated proteins that bind and hydrolyze GTP (for a recent review, see Barbacid, 1987). Genes encoding proteins quite similar in structure have been found across a broad spectrum of eukaryotic species, including the yeasts Saccha- romyces (DeFeo-Jones et al., 1983; Powers et al., 1984) and Schizosacchuromyces (Fukui and Kaziro, 1985), the cellular slime mold Dictyostelium (Reymond et al., 1984), and diptera such as Drosophila (Neuman-Silberberg et al., 1984; Mozer et al., 1985; Schejter and Shilo, 1985). Transducins and G- proteins that regulate cyclic nucleotide metabolism in mammalian cells share a number of characteristics with the ras protein family. G-proteins function as heterotrimers to link receptor proteins at the cytoplasmic membrane to effector activities within the cell (Fleischnab et al., 1986; Hagag et al., 1986). Evidence consistent with a similar role for RAS protein in yeast cells has been presented (Toda et al., 1985). The role of ras proteins in mammalian cells is not known (Birchmeier et al., 1985; Bar-Sagi and Feramisco, 1985).

All ras proteins appear to be targeted to the inner surface of the plasma membrane by posttranslational addition of

* This work was supported in part by Grants GM26020 (to S. C.), A120980 (to J. S.), and CA41086 (to J. R. B.) from the United States Public Health Service and a Grant DMB862102 (to S. C.) from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

City, IA 52242. $ Present address: Dept. of Biochemistry, University of Iowa, Iowa

II Supported by a Fellowship from the American Heart Association, Greater Los Angeles Affiliate.

)I To whom correspondence should be addressed.

palmitate to a conserved cysteine near the carboxyl terminus (Sefton et al., 1982; Shih et al., 1982; Chen et al., 1985; Buss and Sefton, 1986; Fujiyama and Tamanoi, 1986; Buss et al., 1987). The modified cysteine is the 4th residue from the carboxyl terminus, within a conserved cysteine-aliphatic-aliphatic-variable-COOH motif referred to as a CAAX tail. Mutant ras proteins that lack this conserved cysteine fail to be acylated and do not stably associate with membranes (Willumsen et al., 1984; Deschenes and Broach, 1987). The characteristic CAAX tail has been identified in a wide range of proteins involved in growth regulation and signal transduction (Clarke et al., 1988). These include the a and y subunits of most mammalian G-proteins and transducins (Itoh et al., 1986; Beals et al., 1987; Ovchinnikov et al., 1987b; Hurley et al., 1984); several additional GTP-binding proteins such as the rho, ral, YPT, and smg proteins (Madaule and Axel, 1985; Chardin and Tavitian, 1986; Touchot et al., 1987; Lowe et al., 1987; Pizon et al., 1988; Chardin et al., 1988; Kawata et al., 1988); cGMP phosphodiesterase from bovine retina (Ovchin- nikov et al., 1987a); and the nuclear lamins (Clarke et al., 1988).

Fatty acylation has been described in a wide variety of cellular proteins (reviewed by Sefton and Buss, 1987; Schultz et al., 1988). At least two nonoverlapping pathways have been described for the addition of fatty acids to proteins (Sefton and Buss, 1987; McIlhenney et al., 1985; Olsen and Spizz, 1986). One class is characterized by the cotranslational attachment of myristic acid in an amide linkage to amino- terminal glycine residues. Examples include pp60”“ (Sefton et al., 1982), the CAMP-dependent protein kinase (Carr et al., 1982), and some a subunits of mammalian G-proteins (Buss et al., 1987). The second class, which includes the ras proteins and other GTP-binding proteins such as YPTl (Molendar et al., 1988), are posttranslationally modified by addition of palmitate to cysteine. Palmitate is assumed to be linked to cysteine through a thioester bond, although evidence to sup- port this hypothesis is limited.

Several fungal mating factor lipopeptides share with ras proteins the conserved sequences at the site of lipidation (Table I). However, in these peptides farnesyl derivatives are attached to the conserved cysteine via a thioether linkage (Sakagami et al., 1981; Ishibashi et al., 1984; Betz et al., 1987; Akada et al., 1987; Anderegg et al., 1988). Evidence has been presented that these mating factors are further modified by proteolytic cleavage and by methylation on the a-carboxyl of cysteine (Sakagami et al., 1981; Ishibashi et al., 1984; Anderegg et al., 1988). Sequence similarities between the carboxyl terminus of RAS proteins and the mating pheromones have raised the possibility that a common set of modifying enzymes are involved in their processing. This idea is fortified by the observation that certain yeast mutants unable to process RAS proteins also fail to produce functional a-factor mating pher-

11865

11866 Carboxyl Methylation of Yeast RAS TABLE I

Carboxyl-terminal amino acid sequences of ras proteins and fungal sex factors

Sequences are derived from DNA sequences unless stated otherwise. Primary literature for ras sequences are in Barbacid (1987). Human H-ras-1 K-ras-2A

---Ser-Cys-Lys-Cys-Val-Leu-Ser

N-ras ---1le-Lys-Lys-Cys-Ile-Ile-Met ---Gly-Leu-Pro-Cys-Val-Val-Met

Drosophila

Dictyostelium Dras-1 ---Arg-Phe-Lys-Cys-Lys-Met-Leu

Ddras ---Lys-Lys-Gln-Cys-Leu-Ile-Leu S. cereuisiae

RASl RAs2

---Gly-Gly-Cys-Cys-Ile-Ile-Cys - - - G l y - G l y - C y s - C y s - I l e - I l e - S e r

a-Factor" - - -Asp-Pro-Ala -Cys -Val - I l e -Ala Tremellnb TremerogenA-9291-1 ---Ser-Gly-Gly-C& Tremerogen A-10 ---Am-Gly-Tyr-Cys

Rhodotorucine A ---Arg-Asn-Gly-Cys-Thr-Val-Ala R. toruloides'

The terminal three amino acids are cleaved from mature a-factor (Brake et al., 1985; Betz et al., 1987; Anderegg et al., 1988).

The structures of the tremerogens are derived from the protein sequence of the mature factors (Sakagami et al., 1981; Ishibashi et al., 1984). It is not known whether they are synthesized first as longer precursors.

e Structure derived from cDNA sequence (Akada et al., 1987).

omone (Powers et al., 1986; Fujiyama et al., 1987; Tamanoi et al., 1988).

Consistent with the existence of a common processing pathway for mating pheromones and ras proteins, we showed in a previous report that Ha-ras in mammalian cells is methylated by a novel methyltransferase activity (Clarke et al., 1988). However, the site and the chemical nature of this modification was not determined. In the present report we show that yeast RAS2 protein is also methylated and methylation occurs on or near the carboxyl terminus, most likely at the conserved cysteine, as part of complex series of reactions resulting in a fatty acylated RASS protein. The general role of these proteins in membrane-mediated signaling events raises the possibility that protein methylation may function to modulate membrane signaling events in eukaryotic cells.

EXPERIMENTAL PROCEDURES

Strains and Plasmids-Saccharomyces cereuisiae strains used in this study are listed in Table 11. Diploid JR345 was obtained by mating strains BJ2169 and XJB3-1B and strain JR345-6A was a meiotic spore clone from that diploid. Strain JR345-6A was trans- formed with the plasmids YEP-RAS2, YE~-ras2S"'-~~', or YEp- ras20pa1J18 to produce strains JR865, JR866, and JR867, respectively. The structure of YEP-RAS2 is described in detail in Deschenes and Broach (1987). Standard yeast procedures were used for mating, sporulation, and scoring of tetrads (Sherman et al., 1986).

Induction and Immunoprecipitation of RAS2 Protein-Yeast strains were grown in rich medium (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose) or in synthetic medium (2% glucose plus 0.67% yeast nitrogen base without amino acids, supplemented with amino acids, adenine, and uracil as described (Sherman et al., 1986)). For galactose induction, cells were grown at 30 "C in 50 ml of minimal medium with 3% raffinose in place of glucose. At a cell density of approximately 2 X lo7 cells/ml, the culture was made 4% in galactose. After 2 h, cells were harvested by centrifugation and washed with minimal medium lacking methionine. Cells were resus- pended in %oth volume minimal medium lacking methionine containing either 300 pCi of [methyl-3H]AdoMet1 (5-15 Ci/mM; Amersham Corp.) or 300 pCi of [35S]methionine and incubated either for 30 min in the presence of [ methyl-3H]AdoMet or 2 h in the presence of [35S] methionine. Cells were labeled with 35S04 as described by Deshaies and Sheckman (1987). Cells were harvested, washed once in sorbitol

The abbreviations used are: AdoMet, S-adenosyl-L-methionine; SDS, sodium dodecyl sulfate.

buffer (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgC12, 10 mM Tris-HC1, pH 7.4), and lysed by vortexing with glass beads in 1-2 ml of sorbitol buffer containing 1 mM phenylmethylsulfonyl fluoride, 100 units/ml aprotinin, and 1 p M pepstatin (Sigma). Cellular debris was removed by centrifugation far 10 min at 1,500 X g. Aliquots of the supernatant were adjusted to a final concentration of 1% Triton X-100, 0.5% deoxycholate (Sigma), incubated at 0 "C for 30 min, and centrifuged for 5 min at 12,000 X g. The supernatant was analyzed by immunoprecipitation with rat anti-Ha-ras monoclonal antibody, Y13-259 (Oncogene Sciences, Manhasset, NY), as described previously (Furth et al., 1982; Deschenes and Broach, 1987).

Gel Electrophoresis and Vapor-phose Equilibrium Assay for Methyl Esters-Polyacrylamide slab gel electrophoresis was performed by a modification of the method of Laemmli (1970). Sample buffer con- sisted of 1% sodium dodecyl sulfate (SDS), 280 mM 2-mercaptoetha- nol, 62 mM Tris hydrochloride, 5% glycerol, 0.001% bromphenol blue, pH 6.8. Electrophoresis was performed at 20 mA until the dye front reached the separating gel, at which time the voltage was adjusted to 200 V for the remainder of the run (approximately 4 h). Gels were stained for 2 h in Coomassie Blue dye dissolved in 25% isopropyl alcohol and 10% acetic acid (w/v). After destaining overnight in 10% acetic acid (w/v), gels were photographed, and then dried under vacuum at 60 'C. Radiolabeled methyl esters were assayed by the transfer of [3H]methanol from base-hydrolyzed protein methyl esters to scintillation fluid (Stock et al., 1984). Dried gel slices were cut into 0.3-cm slices and mixed with 75 p1 of 1 M NaOH in a 1.5-ml polyethylene microcentrifuge tube. The tube was placed in a 6-ml plastic scintillation vial containing 2.4 ml of scintillation fluid (Li- quiscint, National Diagnostics). After 24 h equilibration at 37 "C, radioactivity that had been transferred to the scintillation fluid was assayed in a liquid scintillation spectrometer.

Cyanogen Bromide Treatment and Carboxypeptidase Digestion of Immunoprecipitated RAS2 Protein-RAS2 protein was immunoprecipitated from extracts of strain JR865 labeled with 35S04 or strain JR830 labeled with [3H]AdoMet. Immunoprecipitated RAS2 protein bound to protein A-Sepharose was washed six times in RIPA (10 mM Tris-HC1, pH 7.4, 0.15 M NaCl, 1% Triton X-100, 1% deoxycholate) and once with 50 mM sodium acetate, pH 5.5. The sample was suspended in 0.3 ml of 50 mM sodium acetate, pH 5.5, and 250 units of carboxypeptidase Y (Calbiochem) was added. At the indicated times, the reaction was stopped by addition of 5 mg of cyanogen bromide in 80% formate. Cyanogen bromide cleavage was performed for 24 h at room temperature. Samples were lyophilized and prepared for electrophoresis as described above.

Synthesis of L-Cysteic Acid Methyl Ester-L-Cysteic acid methyl ester was synthesized exactly as described by Rosowsky et al. (1984), except that the product was crystallized by allowing the methanol/ thionyl chloride mixture to evaporate at room temperature to about 20% of its original volume. Crystals were collected by suction filtration on a sintered glass funnel and washed with diethyl ether. The product was characterized by thin layer chromatography on cellulose plates using a solvent system of n-butano1:acetic acidwater (4:1:1, v/ v/v). The RF of the major ninhydrin-reactive product was 0.31; a small amount (about 5%) of the cysteic acid starting material was also present with an RF value of 0.04.

Performic Acid Oxidation and Enzymatic Digestion of Radiolabeled RAS2 Proteins-RAS2 protein was immunoprecipitated prepara- tively from extracts of approximately 2 X 10' [3H]AdoMet or 3sso4- labeled cells and fractionated on a preparative SDS-polyacrylamide gel. Fractionated protein was transferred to an immobilon membrane by electroblotting and the region containing RAS2 protein was ex- cised. Membrane-bound RAS2 protein was subjected to a series of chemical and enzymatic treatments as described in the following. At each stage of the treatment of methylL3H-labeled RAS2 protein, the presence of radiolabeled methyl esters on the membrane and in the reaction and wash solutions was monitored by assaying transfer of volatile [3H]methanol from base-hydrolyzed protein methyl ester to scintillation fluid by procedure B of Murray and Clarke (1986). The partitioning of 35S-labeled RAS2 protein between membrane and solutions was monitored by direct counting.

A 3 X 0.7-cm sample of RAS2 protein-impregnated membrane was diced into 1 X 1-mm squares and placed in a 7-ml scintillation vial. The membrane was washed twice with 0.5 ml of water by swirling the water over the membrane pieces for at least 30 s and then decanting. The membrane-containing vial was cooled on ice, and performic acid oxidation was performed by a modified procedure of Hirs (1967). Ice- cold performic acid solution (0.5 ml) was added to the membrane- containing vial and allowed to stand at 0 "C for 1 h. The filtrate and

Carboxyl Methylation of Yeast RAS 11867

Strain

XJB3-1B BJ2169 JR345

JR345-6A JR865 JR866 JR867 JR830

TABLE I1 Yeast strains used in this study

Genotype Source

MATa met6 ATCC MATa leu2 urd3 trpl prbl-1122 prc407 pep4-3 E. W. Jones MATaIMATa leu21 + t r p l / + urd31 + his31 +

met61 + prb-11221 + prc4071 + pep4-31 + MATa leu2 trpl ura3 his3 met6 This study MATa leu2 trpl urd3 his3 met6 (YEP-RAS2) This study MATa leu2 trpl urd3 his3 met6 (YEp-ra~2~'"'') This study MATa leu2 trpl urd3 his3 met6 ( Y E p - r a ~ 2 ~ ~ ' ~ ~ ~ ~ ) This study MATa leu2 trpl urd3 his3 pep4::URAS (YEp-RAS2) This study

two or three water washes of the oxidized membrane were lyophilized to dryness in a 1.9-ml polyethylene tube. This material was found to contain greater than 80% of the radiolabeled RAS2 protein and was subjected to enzymatic digestion.

Trypsin (Sigma, Type XI, bovine pancreas, treated with diphenyl carbamyl chloride, 9000 Ne-benzoyl-L-arginine ethyl ester unitslmg protein) was dissolved in 0.1 N HCl to a protein concentration of 10 mg/ml. Digestion buffer (0.5 ml, 0.1 M sodium bicarbonate, 0.1 mM calcium chloride, pH 8.0) and an aliquot of the trypsin solution (7.5 pl, 727.5 units) were added to the tube containing the lyophilized residue from the proceeding performic acid oxidation. The tube was capped and incubated at 37 "C for 1.5 h. An additional aliquot (7.5 pl) of the trypsin solution was added, and incubation was continued at 37 "C for an additional 1.5 h.

Leucine aminopeptidase (Sigma, Type VI, porcine kidney microsomes, lyophilized powder containing approximately 50% protein, 89 L-leucinamide unitslmg protein) was dissolved in water to a protein concentration of 5 mg/ml. Alternatively, leucine aminopeptidase (Sigma, Type IV-S, porcine kidney microsomes, suspension in 3.5 M ammonium sulfate solution, pH 7.7, containing 10 mM magnesium sulfate, 10-20 L-leucinamide unitslmg protein) was used. Following trypsin digestion, an aliquot of leucine aminopeptidase solution (0.45 unit) was added and the solution incubated at 37 "C for 1.5 h. An additional equivalent amount of leucine aminopeptidase was added and the incubation continued for an additional 1.5 h. The digestion was immediately frozen and lyophilized to dryness.

RESULTS

The Yeast RAS2 Protein Is Methyl-esterified in Vivo-In order to examine methylation of RAS protein in yeast, we measured the presence of labile methyl groups in fractionated extracts of strains in which RAS2 protein was overproduced. Protein methylation was assayed by measuring the liberation of [3H]methanol from methyL3H-labeled proteins incubated under mild alkaline conditions. To label RAS2 protein, a methionine auxotroph, strain JR865, harboring a plasmid with a RAS2 insert behind a GAL10 promoter, YEP-RAS2, was labeled with S-adenosyl[ methyl-3H]methionine. Unlike mammalian cells and most bacterial species, yeast readily accumulate exogenous AdoMet (Cherest et al., 1973). Expres- sion of RASS was either induced by growth on galactose or repressed by growth on glucose. The parent strain lacking the plasmid, strain JR345-6A, was labeled under similar conditions. As evident in Fig. 1, a single major peak of protein methyl ester with an apparent molecular weight of 38,000 was observed in cells where RAS2 was expressed at high levels, but not under conditions where RASB was repressed or in strains lacking a high copy RASB expression vector. The identity of the methylated species with RASB was confirmed by immunoprecipitation with a monoclonal anti-ras antibody as described under "Experimental Procedures."

Carboxyl-terminal Mutations Block Methylation of RAS2 Protein-If the yeast RAS2 protein is methylated as part of the maturation of its carboxyl terminus, one might expect that mutations altering this region would block methylation. Accordingly, we measured the extent of methyl esterification

sa0

0

FIG. 1. Detection of methyl esters by vapor-phase equilibrium assay in strains expressing RAS2 protein on galactose- inducible yeast plasmids. Total extracts from [me t l~y l -~H] AdoMet-labeled JR345-6A or JR865 cells were fractionated by electrophoresis on an SDS, 15% polyacrylamide gel. Tracks were sliced into 3-mm gel slices and assayed for base-volatilized radioactivity. Solid circles, strain JR865 (YEP-RASB), after induction with galactose; open circles, strain JR865, after growth on glucose; open triangles, strain JR345-6A (plasmid-free parent strain), after induction with galactose. Gel slice numbering begins at the top of the gel, and the relative migration distance for prestained molecular mass markers is shown in kilodaltons (phosphorylase b, 97.4; bovine serum albumin, 68; ovalbumin, 43; a-chymotrypsinogen, 25.7; P-lactoglobulin, 18.4; cytochrome c, 12.3).

of mutant ras2 proteins containing alterations of the conserved cysteine residue. The high copy galactose-inducible expression vector used to study wild type RAS2 was subjected to oligonucleotide-directed mutagenesis to produce two ras2 variants that lack Cys-319, the conserved CAAX sequence cysteine residue. In one mutant, Cys-319-Ser, a serine codon was substituted for the cysteine codon; in the other, Cys-318- Opal, a nonsense termination codon was introduced just prior to the conserved cysteine residue (Broach et al., 1983; Desch- enes and Broach, 1987). As shown in Fig. 2, a strain containing the wild type RAS2 plasmid yielded rneth~l-~H-labeled RAS2 protein following the labeling regime described above. On the other hand, strains containing either of the mutant ras2 plasmids did not yield a rneth~l-~H-labeled species. These data are consistent with the hypothesis that the site of methyl esterification is at, or near, the carboxyl terminus.

The absence of n~thyl-~H-labeled RASB protein in strains carrying the mutant ras2 plasmid is not the result of dimin- ished production of protein from these plasmids. Results in Fig. 3, showing immunoprecipitation of RAS2 protein from the test strains labeled with [35S]methionine, demonstrate that mutant and wild type proteins accumulate to comparable levels following induction. We note that in SDS-polyacrylamide gel electrophoresis of the immunoprecipitates, wild type

11868 Carboxyl Methylation of Yeast RAS

l2O0 1 200

180 43 25.7 18.4 12.3

160

- 120

- 140

-

100 8 0 - - h ll p.0

, . , I . . , . . I . . . . .

0 1 2 3 4 5 8 7 8 9 10 11 12 13 14 15 16 17 18 19 0 2 4 6 8 10 12 14 18 18 20 22 24 26

FIG. 5. Cyanogen bromide cleavage of methyl-’H-labeled RAS2 protein. Strain JR865 was induced with galactose and labeled with [3H]AdoMet as described under “Experimental Procedures.” RAS2 protein was immunoprecipitated from an extract of the labeled cells and either fractionated directed on an SDS, 17.5% polyacrylamide gel (filled circles) or digested with cyanogen bromide prior to gel fractionation (open circles). Gel slices were assayed for base- volatile radioactivity as described in the legend to Fig. 2. The positions of migration of various molecular mass markers (kDa) are shown.

Gel slice FIG. 2. Detection of methyl esters by vapor-phase equilib-

rium assay in strains with inducible wild type RAS2 and mutant ra9 containing plasmids. Strains JR865 (filled circles), 866 (open circles), and 867 (open triangles) were induced with galactose and labeled with [3H]AdoMet as described under “Experimental Procedures.” Extracts were fractionated by SDS-polyacrylamide gel electrophoresis and the gel processed as described in the legend to Fig. 1.

100-

5 0

25”

0 15 50 45 00 75 90 1 0 5 1

88- ’

” d

25.7-

18.4-

FIG. 3. Galactose-induced expression of wild type and mutant RAS2 proteins. Strains JR865 (lune 1 ), JR866 (lune 2), or JR867 (lane 3) were grown at 30 “C on synthetic complete medium, induced for 2 h with galactose, and then labeled for 2 h with [“SI methionine as described under “Experimental Procedures.” RAS2 protein was immunoprecipitated from cell extracts of the labeled strains and fractionated on an SDS, 15% polyacrylamide gel. Position of migration of molecular weight markers are indicated on the left.

1 !O

TIME (minutes)

FIG. 6. Carboxypeptidase treatment of methyZ-’H-labeled RAS2 protein. Strain JR865 was induced with galactose and labeled with [3H]AdoMet. RAS2 protein was immunoprecipitated from the total cell extracts. The immunoprecipitate was treated with carboxypeptidase for the times indicated, resolved on SDS-polyacrylamide gel electrophoresis, and the base volatile radioactivity in the 38-kDa RASP peak was determined.

1 2 9 4 s

RAS2 protein resolves into two species migrating at approximately 38,000 molecular weight. Pulse-chase experiments with [35S]methionine indicate that the slower migrating form is a precursor of the faster migrating species (data not shown). We have previously shown that only the faster form is associated with the membrane fraction (Deschenes and Broach, 1987). The rm2Ser-319 protein comigrates with the precursor, while the r a ~ 2 ~ ~ ~ - ~ ~ ~ protein comigrates with the mature form of RAS2. Neither mutant is palmitoylated, and neither is localized to the membrane fraction (Deschenes and Broach, 1987). Comigration of the Cys-318-Opal mutant with the mature form of RAS2 protein is consistent with additional modification involving proteolysis of the CAAX tail.

RAS2 Protein Is Methyl-esterified Near Its Carboxyl Ter- minus-We used cyanogen bromide (CNBr) digestion of labeled RAS protein to localize further the site of methylation. Cleavage at the six internal methionine residues in RAS2 should yield seven fragments, the largest of which extends 109 residues from Met-213 to the carboxyl terminus. Since CNBr cleavage converts methionine to homoserine, the only

18.4,

18.3

FIG. 4. Digestion of S6S04-labeled RAS2 with cyanogen bromide and carboxypeptidase Y. Strain JR830 was induced with galactose and labeled with 35S04 as described under “Experimental Procedures.” RASP protein was immunoprecipitated from the labeled cell extract and either fractionated directly by electrophoresis on an SDS, 17.5% polyacrylamide gel (lune 1 ) or treated with carboxypeptidase Y for 0 (lane 2) , 15 (lane 3 ) , 30 (lane 4) , or 60 (lane 5) min prior to cyanogen bromide cleavage and gel fractionation.


2250

1750

1250

75c

25C

C

A : Sephadex G-15 3.0

I . . . . b . . . . I . . I 30 40 50

Fraction Number

280 1 B: Thin-layer Electrophoresis

l o [ CAME 1 200

- 4 - 2 0 2 4

Migration from Origin (cm)

C : Ion-exchange 110 - - 0.9

Fraction Number

FIG. 7. Isolation and characterization of a 3H-methylated product of enzymatically digested yeast RAS2 protein. A prep- aration of immobilon membrane containing RAS2 protein labeled with [3H]methyl groups was subjected to performic acid oxidation,

sulfur remaining after cleavage is in cysteine residues. RAS2 protein contains only 2 cysteine residues, both of which are located within the carboxyl-terminal cyanogen bromide fragment. Thus, only the carboxyl-terminal CNBr fragment from Y3-labeled RAS2 should be radioactive. When we digested 35S-labeled RAS2 protein with cyanogen bromide, two 35S- labeled peptides of apparent molecular mass 22 and 12 kDa were observed (Fig. 4, lane 2). The 12-kDa peptide is most likely the COOH-terminal fragment, and the 22-kDa species may be a partial fragmentation product. Cyanogen bromide cleavage of [3H]AdoMet-labeled RAS2 resulted in peptides with the same apparent mobility as the 35S-labeled RAS2 cyanogen bromide fragments (Fig. 5 ) . These results indicate that RAS2 protein is methylated somewhere between Met- 213 and the carboxyl terminus.

We next digested each of these labeled RAS2 proteins with carboxypeptidase Y and compared the rate of loss of radioactivity. The rate of loss of [35S]cysteine was estimated by scanning lanes 2-5 of Fig. 4. A t1/, of approximately 10 min was observed. When the same experiment was performed with the methyL3H-RAS2 material and radioactivity was plotted against time of carboxypeptidase Y treatment (Fig. 6), a t1/, of approximately 7 min was obtained. We conclude from these experiments that the methylation of RAS2 occurs in the carboxyl-terminal cyanogen bromide fragment in close prox- imity to the carboxyl-terminal cysteine.

RAS2 Is Methyl-esterified on a Cysteine Residue-Since the labile methyl group in RAS2 protein is located near the carboxyl terminus, we asked whether it was actually located on a cysteine residue. To accomplish this, we used a combi- nation of enzymatic digestions to reduce 35S- or 3H-labeled RAS2 protein to its constituent amino acids in a manner that

followed by trypsin digestion, and finally leucine aminopeptidase digestion as described under “Experimental Procedures.” Panel A, lyophilized enzymatic digest (about 3000 cpm) was analyzed by gel filtration chromatography. Prior to application on the column, cysteic acid methyl ester (CAME) (1 pmol) was added to the enzymatic digest as an amino acid standard. The sample and standard were applied in 0.1 N acetic acid (approximately, volume 0.4 ml) to a 1.5 X 81-cm gel filtration column consisting of Sephadex G-15 resin. The mixture was eluted with 0.1 N acetic acid, and 6-min fractions were collected at room temperature with a flow rate of 0.34 ml/min. Fractions 30-55 were lyophilized individually, and the residues were taken up into 0.10 ml of water. Filled circles, ninhydrin analysis (Moore, 1968) of 25-pl samples of rehydrated material from individual fractions; open circles, total radioactivity in 25-pl samples of rehydrated material from individual fractions. Panel B, the charge of the rnethyl-3H-labeled material in a portion of fraction 48 from G-15 gel filtration chromatography was determined by thin layer electrophoresis. The sample was applied to the center (origin) of a 20-cm cellulose sheet (Eastman Kodak No. 13254). Electrophoresis was performed in a 225:25:1 water:pyridine:acetic acid (pH 6.5) buffer at 400 V for 20 min at room temperature. Location of base-labile volatile counts on the chromatogram were determined by slicing it into 0.5- cm strips and treating them in the same manner as gel electrophoresis slices in Fig. 2 and “Experimental Procedures.” The position of migration of cysteic acid methyl ester (CAME) was determined using ninhydrin spray. Panel C , an aliquot of fraction 48 of the G-15 gel filtration chromatography shown in Panel A was subsequently analyzed by isocratic cation exchange chromatography. Prior to application on the column, additional cysteic acid methyl ester (1 pmol) was added to the sample as a standard. The sample and standard were applied to a cation exchange column (0.9 X 54 cm) consisting of sulfonated polystyrene amino acid analysis resin (Beckman AA-15) equilibrated in 0.2 M sodium citrate buffer, pH 3.25 (Pierce, pHix) at 56 “C. One-minute fractions were collected at a flow rate of 1.2 ml/ min, and aliquots (50 pl) were subjected to ninhydrin analysis to locate the cysteic acid methyl ester standard (filled circles) as described by Moore (1968). The remaining fraction (1.0 ml) was counted in 10 ml of scintillation fluid for radioactivity (open circles). Under these conditions, aspartic acid elutes at 35-36 min.


50 4 1 A : Control 2’o

lo'""""^^' 0 10 20 30 40 50

0.0

Fraction Number

6o t B : Base Treated

10 0 10 20 30 40 50

Fraction Number

FIG. 8. Isolation and characterization of a ‘%-methylated product of enzymatically digested yeast RAS2 protein. A prep- aration of immobilon membrane containing immunoprecipitated RASS protein labeled with [35S]sulfate was subjected to performic acid oxidation, followed by trypsin digestion, and finally leucine aminopeptidase digestion as described under “Experimental Proce- dures.” The lyophilized enzymatic digest was dissolved in 0.1 ml of water, and the solution was divided into two equivalent portions. Panel A, one portion (50 pl) of the enzymatic digestion was mixed with cysteic acid methyl ester (1 pmol) and subjected to isocratic cation exchange chromatography as described in Fig. 7. Ninhydrin (filled circles) and total counts analysis (open circles) were performed as described in Fig. 7. The elution time determined in a separate experiment for methionine sulfone (40 min) is shown for reference. Panel B, the remaining portion (50 pl) of the enzymatic digest was treated with sodium hydroxide (0.1 mmol) for 24 h at room temperature. The solution was neutralized with dilute hydrochloric acid and lyophilized to dryness. The residue was subjected to isocratic cation exchange chromatography as described in Fig. 7.

would not cleave a methyl ester bond. We then asked if the amino acid residue containing the labile [3H]methyl group comigrated under a variety of chromatographic conditions with a similarly derived 36S-labeled amino acid.

The results of this analysis are presented in Figs. 7 and 8. RASS protein labeled with [3H]AdoMet was immunoprecipitated, gel fractionated, transferred to a polyvinylidene diflu- oride membrane and then digested with trypsin and leucine aminopeptidase. Prior to digestion, the material was treated with performic acid to oxidize cysteine residues to cysteic acid residues and methionine residues to methionine sulfone residues and to remove thioester-linked palmitic acid. These

treatments liberated a small molecular weight, hydrophilic, 3H-labeled species that chromatographed as a single peak on a Sephadex G-15 column in the same position expected for free amino acids and eluted as a single peak at 38 min near aspartic acid during isocratic cation exchange column chromatography. Similar treatment of 35S-labeled RAS2 protein yielded several labeled species, as would be expected, one of which migrated on the isocratic ion exchange column at the same position as the 3H-labeled compound. We confirmed that the 35S-labeled species migrating at 38 min carried an alkali-labile methyl group by demonstrating that the peak was eliminated by mild base treatment of the sample prior to chromatography (Fig. 8). We conclude from these results that the labile methyl group is present on a sulfur-containing amino acid.

Although our results suggest that RAS2 is modified by methyl esterification of a cysteine residue, the structure of the final modified product appeared to be more complex than we had originally anticipated. If, as we expected, a carboxyl- terminal cysteine residue is modified by methyl esterification of the a-carboxy group, then chemical and enzymatic treatment of RASS protein as described above should have yielded cysteic acid methyl ester. To test this, we synthesized cysteic acid methyl ester and compared its chromatographic behavior with that of the labile methyl-containing amino acid derived from RAS2 protein. Although the two species comigrate in gel filtration chromatography and in thin layer electrophoresis, they are readily separable by isocratic cation exchange chromatography (Fig. 7). Thus, although the methyl-modified amino acid in RAS2 is most likely a carboxyl-terminal cysteine, this residue appears to undergo additional and currently undefined modification at the side chain sulfur.

DISCUSSION

A growing list of proteins differentially associate with membranes as a consequence of posttranslational lipidation. Lip- idation of yeast RAS protein occurs at a cysteine residue that, in the nascent polypeptide, is part of a CAAX tail conserved among known ras-like proteins (Willumsen et al., 1984; Deschenes and Broach, 1987). Failure to palmitylate this cysteine precludes membrane association and interdicts RAS- dependent activity, both in yeast and mammalian ras proteins (Willumsen et al., 1984; Deschenes and Broach, 1987).

Several lines of evidence suggest that RAS2 protein carboxyl-terminal processing involves several additional steps. First, the apparent molecular mass of RAS2 protein decreases by 0.5 kDa in conjunction with palmitoylation and membrane attachment. This is true as well of mammalian ras proteins (Shih et al., 1982). This shift could be explained by proteolytic cleavage of the carboxyl-terminal three amino acids. Second, precedent for multistepped maturation pathway for lipid ad- ditions to proteins is provided by yeast a-mating pheromone. The yeast a-mating factor, which has the sequence Cys-Val- Ile-Ala at its carboxyl terminus (Brake et al., 1985), is proteo- lytically processed to remove the 3 terminal residues (Ander- egg et al., 1988). The resulting carboxyl-terminal cysteine is further modified by ether linkage of lipid to the free thiol and methyl esterification of the a-carboxyl (Anderegg et al., 1988). The product of a locus in yeast, variously termed DPRl or RAMl, is necessary for processing a-factor as well as RAS protein (Powers et al., 1986; Fujiyama et al., 1987). Accord- ingly, RAS processing should involve some steps in common with a-factor processing distinct from acylation. Accumula- tion of full-length RAS2 precursor protein in dprllraml strains could argue that this gene promotes proteolytic processing. Finally, we recently reported that the mammalian ras

Carboxyl Methylation of Yeast RAS 1187 1

protein contains a labile methyl group, which, by analogy to the Tremella mating factors, we speculated occurs on the a- carboxyl of cysteine (Clarke et al., 1988). In this report we have investigated the modification of yeast RAS2 protein and have found that yeast RAS2 protein is also methylated and that the conserved cysteine is involved. On the basis of available peptide chemistry, the effects of ras2 mutations, and by analogy to yeast mating factors, this modification is most likely a methyl ester of the a-carboxyl group of cysteine. This site would be available only upon proteolytic removal of the carboxyl-terminal 3 residues from RAS protein precursor. Thus, RAS protein appears to undergo carboxyl-terminal methyl esterification and proteolysis, in addition to acylation.

The evidence that the base-volatilized radioactivity found in the RAS protein are derived from a carboxyl-terminal cysteine is as follows. First, no methylation is observed in RAS2 proteins in which the carboxyl terminus has been altered by either a point mutation in the conserved cysteine or a deletion that ablates the two cysteines and the distal three amino acids. Since these mutant proteins are expressed at normal levels and are otherwise functional, the absence of methylation argues strongly that the methylation normally occurs on the conserved cysteine. Second, cyanogen bromide fractionation and carboxypeptidase digestion kinetics localize the methylated group at or near the carboxyl end of the protein. Third, by chemical analysis of the enzymatically digested 3H-RAS2 protein we have ruled out the possibility that the methylated amino acid is serine or isoleucine, the other amino acids near the carboxyl terminus. Finally, the labeled amino acid residue from 3sS- and 3H-RAS2 digestions copurify, indicating that the methylated and sulfate-labeled species are identical.

Direct chemical identification of modified cysteine residue has proved to be difficult. Although it has previously been suggested that palmitate is attached to cysteine directly via a thioester linkage, this has never been directly demonstrated. We suspect that the chemistry of lipid modification may be more complex and that palmitate may be linked to the thiol group through an intermediate moiety. One possibility is that modification is an acylated glyceryl cysteine moiety with a thioether linkage to the cysteine side chain. This type of fatty acylation of a cysteine side chain has been shown to occur in the Escherichia coli murein lipoprotein (Hantke and Braun, 1973). Another possibility is that the carboxyl-terminal cysteine is isoprenylated, exhibiting a chemistry similar to a- factor. The methyl-esterified product we observe could be formed by rearrangement of this modified group. Acylation could occur at internal cysteines as secondary modifications associated with the process of membrane localization.

Our results are consistent with a model for the processing of RAS proteins that involves proteolytic cleavage, lipidation, and methylation (Fig. 9). Presently we do not know the order of the modification steps. Pulse-chase and subcellular fractionation experiments in yeast indicate that cleavage precedes fatty acylation (Fujiyama et al., 1987; Tamanoi et al., 1988). In contrast, a palmitylated tetrapeptide derived from the carboxyl terminus of mammalian Ha-ras has been detected (Chen et al., 1985), suggesting that palmitylation can precede cleavage. The low recovery of the tetrapeptide in that study suggests that this may be a minor product in the processing pathway and that normally maturation proceeds by cleavage followed by palmitylation. This point remains to be addressed.

We do not yet know what role methylation plays in RAS function. The presence of reactive Cys-318 and Cys-319 thiol- ate anions and the Cys-319 a-carboxylate raises the possibility of complex rearrangements. The methylation reaction may

FIG. 9. A model for RAS2 protein processing in yeast. RAS2 protein is initially synthesized as a soluble 39-kDa protein with an intact carboxyl-terminal sequence ending in CIIS. Several enzymatic steps may then be required to process the carboxyl terminus. The results of pulse-chase experiments suggest that proteolytic cleavage removes the 3 terminal amino acid residues (step I); methylation (step 2 ) and lipidation (step 4) then occur on the resulting carboxyl- terminal cysteine. The chemical group and type of linkage of the lipid is still in question and is designated only as R in the model. The order of formation of processing intermediates (steps 2-5) has not been established.

provide an important mechanism to regulate these rearrangements by blocking the carboxylate anion. In addition, blocking the carboxylate and therefore decreasing the hydro- philicity might facilitate interaction of this region of the molecule with the membrane where the reactions may be carried out. The possible regulatory role of methylation is emphasized by the observation that in cell-free extracts derived from macrophages, a protein, M, = 25,000, is carboxy- methylated in a GTP-dependent fashion (Backlund and Ak- samit, 1988). Presumably this protein is a member of the ras superfamily, and there is a link between GTP binding and carboxyl methylation.

Two types of protein carboxymethyltransferases have previously been described (Clarke, 1985). In bacteria, membrane chemoreceptor-transducer proteins are reversibly methylated at glutamate residues, and in mammalian cells as well as in bacteria abnormal aspartate residues (D-ASP or L-isoAsp) side chain carboxyl groups are methylated in damaged or aged proteins. Thus, whereas in bacteria carboxyl methylation provides an important mechanism for regulating the activities of membrane receptor proteins (Stock and Stock, 1987), in mammalian cells it has appeared to function primarily as a mechanism for protein degradation and/or repair (Clarke, 1988). The existence of a third class of methyltransferases is suggested by the Tremellu sex factors (Sakagami et al., 1981; Ishibashi et al., 1984), the S. cerevisiae a-mating factor (An- deregg et al., 1988), and RAS proteins. If the methylation of RAS proteins controls membrane attachment, then methylation may play a role in eukaryotic signal transduction.

In mammalian cells, both palmitylation and carboxyl methylation appear to be reversible modifications (Clarke, 1985; Chelsky et al., 1985; Staufenbiel, 1988). A particularly intrigu- ing example is the cell cycle-dependent carboxyl methylation of lamin B (Chelsky et al., 1987), which apparently is methylated in intact nuclei and demethylated during mitosis. Per- haps ras proteins are continuously cycled in and out of the membrane by a sequence of reactions including lipidation, methyltransferase, methylesterase, and lipase activities. Any of these steps could play an important role in regulating ras activity. One can imagine a ras modification cycle that follows the cell cycle just as the lamin methylation cycle is coordi- nated with the cycle of nuclear replication. It is interesting in


this regard that the unmethylated derivative of the Tremella mating factor is 200 times less active than the methylated form (Ishibashi et al., 1984) and that demethylation of S. cereukiae a-factor abolishes activity (Anderegg, 1988).

We consistently observed a set of proteins in yeast with a mobility of 20-25 kDa that have base labile methyl groups (see Fig. 1). The identity of the protein, or proteins, in this molecular weight range is currently unknown, but recent results suggest that a number of proteins may undergo similar modifications. In mammalian cells two proteins other than rm are known to be methylated the cGMP phosphodiesterase involved in visual signal transduction in retina (Swanson and Applebury, 1983) and nuclear lamin B (Chelsky et al., 1987). Lamin B appears to be isoprenylated as well (Beck et al., 1988). The conserved CAAX sequence is found at the carboxyl end of a number of yeast proteins other than RAS proteins. Examples include RHO1 and RH02, two proteins that exhibit homology to GTP-binding proteins (Madaule et al., 1987). In another case, a yeast GTP-binding protein encoded by YPT, has been described that is palmitylated, even though it ends in cysteine without the CAAX consensus sequence. Similarly, SEC4 protein, a membrane-localized, ras-like protein, termi- nates in a cysteine residue (Salminen and Novick, 1987; Gout et al., 1988). It is tempting to speculate that the carboxyl termini of YPT and SEC4 proteins represents the cleaved form of the CAAX sequence. In this regard, an altered YPTl protein carrying the RAS2 carboxyl terminus is normally processed (Molendar et al., 1988). We anticipate that some or all of these proteins may also be methyl-esterified and the peak of proteins at 25 kDa with labile methyl groups represents these and other as yet unidentified GTP-binding proteins. Based on our work on RAS proteins and the examples of proteins with homologous carboxyl termini, we propose that posttranslational proteolytic cleavage, lipidation, and methylation may be used by the cell as a general mechanism to target proteins to the membrane. If lipidation and methylation are reversible then these steps might be utilized by the cell to modulate the activity of a whole family of membrane-associated proteins.

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